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• W2792103163 abstract "•Study of global phosphoproteome response after activation of different RTKs•Shp-2 is highly phosphorylated upon Pdgf, but not Fgf-2, stimulation•Application of an allosteric Shp-2 inhibitor reveals phosphotyrosine targets of Shp-2•Pdgf-receptor-dependent cell migration is controlled by Shp-2 Despite its low cellular abundance, phosphotyrosine (pTyr) regulates numerous cell signaling pathways in health and disease. We applied comprehensive phosphoproteomics to unravel differential regulators of receptor tyrosine kinase (RTK)-initiated signaling networks upon activation by Pdgf-ββ, Fgf-2, or Igf-1 and identified more than 40,000 phosphorylation sites, including many phosphotyrosine sites without additional enrichment. The analysis revealed RTK-specific regulation of hundreds of pTyr sites on key signaling molecules. We found the tyrosine phosphatase Shp-2 to be the master regulator of Pdgfr pTyr signaling. Application of a recently introduced allosteric Shp-2 inhibitor revealed global regulation of the Pdgf-dependent tyrosine phosphoproteome, which significantly impaired cell migration. In addition, we present a list of hundreds of Shp-2-dependent targets and putative substrates, including Rasa1 and Cortactin with increased pTyr and Gab1 and Erk1/2 with decreased pTyr. Our study demonstrates that large-scale quantitative phosphoproteomics can precisely dissect tightly regulated kinase-phosphatase signaling networks. Despite its low cellular abundance, phosphotyrosine (pTyr) regulates numerous cell signaling pathways in health and disease. We applied comprehensive phosphoproteomics to unravel differential regulators of receptor tyrosine kinase (RTK)-initiated signaling networks upon activation by Pdgf-ββ, Fgf-2, or Igf-1 and identified more than 40,000 phosphorylation sites, including many phosphotyrosine sites without additional enrichment. The analysis revealed RTK-specific regulation of hundreds of pTyr sites on key signaling molecules. We found the tyrosine phosphatase Shp-2 to be the master regulator of Pdgfr pTyr signaling. Application of a recently introduced allosteric Shp-2 inhibitor revealed global regulation of the Pdgf-dependent tyrosine phosphoproteome, which significantly impaired cell migration. In addition, we present a list of hundreds of Shp-2-dependent targets and putative substrates, including Rasa1 and Cortactin with increased pTyr and Gab1 and Erk1/2 with decreased pTyr. Our study demonstrates that large-scale quantitative phosphoproteomics can precisely dissect tightly regulated kinase-phosphatase signaling networks. With advances in proteomic technologies, it is possible to comprehensively analyze human cell proteomes on par with next-generation genomic sequencing (Bekker-Jensen et al., 2017Bekker-Jensen D.B. Kelstrup C.D. Batth T.S. Larsen S.C. Haldrup C. Bramsen J.B. Sørensen K.D. Høyer S. Ørntoft T.F. Andersen C.L. et al.An optimized shotgun strategy for the rapid generation of comprehensive human proteomes.Cell Syst. 2017; 4: 587-599.e4Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, Kim et al., 2014Kim M.-S. Pinto S.M. Getnet D. Nirujogi R.S. Manda S.S. Chaerkady R. Madugundu A.K. Kelkar D.S. Isserlin R. Jain S. et al.A draft map of the human proteome.Nature. 2014; 509: 575-581Crossref PubMed Scopus (1494) Google Scholar). However, routine analysis of post-translational modifications (PTMs) remains challenging due to their generally low cellular abundance and dynamic nature. Above all, site-specific protein phosphorylation on serine, threonine, and tyrosine residues is of great importance in eukaryotic cells due to their crucial role in rapidly regulating essentially all intracellular signaling networks (Olsen et al., 2006Olsen J.V. Blagoev B. Gnad F. Macek B. Kumar C. Mortensen P. Mann M. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.Cell. 2006; 127: 635-648Abstract Full Text Full Text PDF PubMed Scopus (2810) Google Scholar). Protein phosphorylation is tightly regulated in healthy cells by the opposing action of protein kinases and phosphatases, which are the enzymes that write and erase this modification, respectively (Ubersax and Ferrell, 2007Ubersax J.A. Ferrell Jr., J.E. Mechanisms of specificity in protein phosphorylation.Nat. Rev. Mol. Cell Biol. 2007; 8: 530-541Crossref PubMed Scopus (995) Google Scholar). Phosphotyrosine (pTyr) in particular remains elusive in its detection due to its highly transient and regulatory nature, which leads to overall lower phosphorylation stoichiometry and cellular abundance relative to its phosphoserine (pSer) and phosphothreonine (pThr) counterparts, which are readily observed in global phosphoproteomic experiments (Sharma et al., 2014Sharma K. D’Souza R.C.J. Tyanova S. Schaab C. Wiśniewski J.R. Cox J. Mann M. Ultradeep human phosphoproteome reveals a distinct regulatory nature of Tyr and Ser/Thr-based signaling.Cell Rep. 2014; 8: 1583-1594Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar). As a result, many large-scale cell signaling studies may inadvertently omit pTyr analysis and thereby overlook a vital layer of information regulating cell signaling and outcome. Obtaining greater molecular understanding of pTyr-driven signaling (alongside pSer and pThr) is the foundation for understanding signaling networks such as those initiated by receptor tyrosine kinases (RTKs), which are able to receive extracellular signals and relay them inside the cell plasma membrane, ultimately affecting cell fate, such as survival, differentiation, proliferation, and migration (Lemmon and Schlessinger, 2010Lemmon M.A. Schlessinger J. Cell signaling by receptor tyrosine kinases.Cell. 2010; 141: 1117-1134Abstract Full Text Full Text PDF PubMed Scopus (3106) Google Scholar). Consequently, RTKs and proteins involved in their downstream signaling tend to be highly oncogenic and thus the foremost drug targets in various cancers (Koytiger et al., 2013Koytiger G. Kaushansky A. Gordus A. Rush J. Sorger P.K. MacBeath G. Phosphotyrosine signaling proteins that drive oncogenesis tend to be highly interconnected.Mol. Cell. Proteomics. 2013; 12: 1204-1213Crossref PubMed Scopus (30) Google Scholar). To address this challenge, we developed a streamlined phosphoproteomic framework based on high-pH reversed-phase chromatography (Wang et al., 2011Wang Y. Yang F. Gritsenko M.A. Wang Y. Clauss T. Liu T. Shen Y. Monroe M.E. Lopez-Ferrer D. Reno T. et al.Reversed-phase chromatography with multiple fraction concatenation strategy for proteome profiling of human MCF10A cells.Proteomics. 2011; 11: 2019-2026Crossref PubMed Scopus (397) Google Scholar) with automatic fraction concatenation followed by sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. This enabled global detection of pSer, pThr, and pTyr sites in a single analysis without the need for phosphotyrosine-specific antibody pull-downs. By primarily using phosphotyrosine site regulation as the proxy of early signaling events, the initial analysis allowed us to observe distinct activation of numerous signaling pathways with RTK ligand treatment. We found platelet-derived growth factor receptor (Pdgfr) (α and β subunits) to be a potent activator of most downstream signaling pathways. We observed that Shp-2 acts as a master regulator of the mitogen-activated protein kinase/extracellular-regulated kinase (Mapk/Erk) pathway for Pdgfr, and through the application of an allosteric Shp-2 phosphatase inhibitor, in combination with quantitative phosphoproteomics, our analysis revealed global rewiring of the tyrosine phosphoproteome in a Pdgf-ββ-dependent manner. This dataset also provides a list of putative Shp-2 substrates, which include proteins regulating downstream RTK signaling pathways important for cellular outcomes such as proliferation and migration. Collectively, our investigation may increase our mechanistic insight into RTK signal propagation and phosphatase action. We employed an optimized phosphoproteomic workflow based on label-free quantitation to unravel regulators of downstream RTK signaling networks mediated by three activated RTKs. We investigated the global phosphosignaling response initiated by three RTKs in mice fibroblast NIH/3T3 cells after stimulation with their respective canonical ligands: platelet-derived growth factor beta receptor (Pdgf-ββ ligand), fibroblast growth factor receptor 1 (Fgf-2 ligand), and insulin-like growth factor 1 receptor (Igf-1 ligand), which were selected due to their abundance (Figure S1A). Their ligands induced a downstream phosphorylation response, evidenced by the activation and phosphorylation of Mapk/Erk (Figure S1B). We performed time-resolved quantitative phosphoproteomics to study the dynamic processes underlying the activation of RTK signal transduction. Samples were collected after 3 and 15 min of ligand stimulation, followed by efficient lysis and protein extraction, cysteine reduction, and alkylation in one step as previously reported (Figure 1A) (Jersie-Christensen et al., 2016Jersie-Christensen R.R. Sultan A. Olsen J.V. Simple and Reproducible Sample Preparation for Single-Shot Phosphoproteomics with High Sensitivity.Methods Mol. Biol. 2016; 1355: 251-260Crossref PubMed Scopus (25) Google Scholar, Poulsen et al., 2013Poulsen J.W. Madsen C.T. Young C. Poulsen F.M. Nielsen M.L. Using guanidine-hydrochloride for fast and efficient protein digestion and single-step affinity-purification mass spectrometry.J. Proteome Res. 2013; 12: 1020-1030Crossref PubMed Scopus (32) Google Scholar). We have previously demonstrated an efficient strategy for offline fractionation of phosphopeptides based on high-pH fractionation, which enabled us to identify a large number of tyrosine-phosphorylated peptides, similar to what is typically observed with the extensive use of pan-anti-pTyr antibodies (Batth et al., 2014Batth T.S. Francavilla C. Olsen J.V. Off-line high-pH reversed-phase fractionation for in-depth phosphoproteomics.J. Proteome Res. 2014; 13: 6176-6186Crossref PubMed Scopus (203) Google Scholar). To establish a streamlined and comprehensive phosphoproteomic workflow, we automated the offline fractionation and concatenation (AUTOCON) steps before mass spectrometry (MS) analysis (Movie S1), which allowed us to process samples in batch mode and increase our throughput and reproducibility (Figure 1B). The phosphoproteomic workflow enabled the identification of 41,289 phosphorylation sites covering more than 7,000 phosphoproteins. We applied a strict quality filter. The resulting phosphosite intensities covered a large dynamic range, more than 6 orders of magnitude in each experiment, and 29,027 phosphorylation sites were quantifiable across conditions (Table S1). Analysis of the phosphorylated amino acid distribution showed that 84% of phosphorylation sites were on serine, whereas the 13% were threonine phosphorylation sites (Figure 1C). In addition, 851 (3%) of the total quantifiable phosphorylation sites belonged to tyrosine-containing peptides (Figure 1C), pinpointing a major strength of our workflow in achieving high pTyr coverage without pretreating cells with pervanadate. Phosphotyrosine sites were roughly 2-fold lower in abundance compared to pSer and pThr (Figure S2A), which underscores the general challenges in detecting them by MS. Our workflow led to an efficient accumulation of phosphotyrosine-containing peptides over the 12 high-pH fractions (Figure S2B), resulting in the identification of pTyr sites on more than 500 proteins enriched for RTK signaling, kinase activity, and protein binding and inclined to be localized at the plasma membrane and cytoplasm (Figure S2C). These data demonstrate the streamlined and comprehensive phosphoproteomic workflow encompassing serine, threonine, and pTyr. We analyzed the ligand-induced distribution of the quantified pTyr sites to gain insights into pTyr signaling upon differential RTK stimulation. Specifically, the proportional response in the pTyr site intensities (ternary plot) (Figure 2) distinguished the stimulating condition under which the sites are distributed relative to one another. Activated Pdgfr was the most potent regulator of pTyr sites when compared to Fgfr1 and Igf1r, as shown by a large cluster of pTyr sites at the top of the ternary plot, indicating higher relative response. Density estimation of all phosphorylation sites under the three RTK stimulation conditions indicated that this phenomenon was not global and was limited only to pTyr, because similar distributions were not observed for pSer and pThr sites (Figure S3). We found 21 pTyr sites on the cytoplasmic tail near the C terminus of Pdgfr-α (11) and Pdgfr-β (10) subunits (Heldin and Lennartsson, 2013Heldin C.-H. Lennartsson J. Structural and functional properties of platelet-derived growth factor and stem cell factor receptors.Cold Spring Harb. Perspect. Biol. 2013; 5: a009100Crossref PubMed Scopus (96) Google Scholar) to be exclusively detected upon Pdgf-ββ stimulation. In addition, we found six pTyr sites on Fgfr1 and fibroblast growth factor receptor 2 (Fgfr2) when stimulated with Fgf-2 (Figure 2; Table S1) (Furdui et al., 2006Furdui C.M. Lew E.D. Schlessinger J. Anderson K.S. Autophosphorylation of FGFR1 kinase is mediated by a sequential and precisely ordered reaction.Mol. Cell. 2006; 21: 711-717Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Sites on canonical adaptor molecules such as Grb2 (pY209), Frs2 (pY306), and Irs2 (pY671) were also identified, indicating their preferential binding to activated Pdgfr, Fgfr1, and Igf1r, respectively (Figure 2). The analysis demonstrated the sensitivity of our workflow for screening pTyr sites without the need for pan-anti-pTyr antibody-based enrichment and comparable to state-of-the-art, antibody-based, phosphotyrosine-focused studies (Abe et al., 2017Abe Y. Nagano M. Tada A. Adachi J. Tomonaga T. Deep phosphotyrosine proteomics by optimization of phosphotyrosine enrichment and MS/MS parameters.J. Proteome Res. 2017; 16: 1077-1086Crossref PubMed Scopus (31) Google Scholar). The number of tyrosine sites reported in this study were roughly 6-fold higher than those reported with the EasyPhos method, which was used to study insulin signaling (another RTK) in vivo over several time points (Humphrey et al., 2015Humphrey S.J. Azimifar S.B. Mann M. High-throughput phosphoproteomics reveals in vivo insulin signaling dynamics.Nat. Biotechnol. 2015; 33: 990-995Crossref PubMed Scopus (310) Google Scholar). The distinct activation of pTyr sites upon ligand stimulation suggests that the response initiated by each ligand-RTK pair differentially regulate downstream signaling pathways (Francavilla et al., 2016Francavilla C. Papetti M. Rigbolt K.T.G. Pedersen A.-K. Sigurdsson J.O. Cazzamali G. Karemore G. Blagoev B. Olsen J.V. Multilayered proteomics reveals molecular switches dictating ligand-dependent EGFR trafficking.Nat. Struct. Mol. Biol. 2016; 23: 608-618Crossref PubMed Scopus (68) Google Scholar). We visualized their temporal changes as quantitative dot blots (Figure 3A) and validated our results by western blot analysis and quantification (Figure 3B; Figure S4). We found Pdgf-ββ and Fgf-2 stimulation induced phosphorylation of the activation loop residues pT203 and pY205 on Mapk/Erk1 (Figure 3). However, we observed that Pdgf-ββ exclusively activated Pik3/Akt (i.e., Pik3ca pY508 and Akt1 pS473), as well as Jak/Stat (i.e., Stat3 pY705) and Plc-γ (i.e., Plcg1 pY783) pathways. Conversely, Fgf-2-treated cells pinpointed a preferential phosphorylation of Y614 and Y615 on focal adhesion kinase 1 (Ptb2 or Fak1) (Figure 3B). Surprisingly, Pdgf-ββ-stimulated cells displayed a reduction in the basal level of phosphorylation of Y614 and Y615 on Fak1 at both time points despite the relatively high pTyr response seen upon Pdgf-ββ stimulation. Because Igf-1 stimulation did not significantly induce activation of major signaling pathways, the remainder of this study was focused on the comparative analysis of the signaling responses mediated by Pdgfr and Fgfr1 activation. Collectively, these data support the comprehensive phosphorylation workflow to investigate dynamic RTK signaling pathways. We hypothesized the presence of a Pdgf-ββ-dependent regulatory mechanism leading to Y614 and Y615 dephosphorylation on Fak1. We speculated that activated tyrosine phosphatases might play a role in mediating this and confirmed the possibility with global inhibition of tyrosine phosphatase activity with pervanadate (Figure S5A). Thus, we examined phosphatases exclusively tyrosine phosphorylated upon Pdgfr activation. We found protein tyrosine phosphate non-receptor type 11 (Shp-2 encoded by PTPN11) to be a putative candidate, because it displayed pronounced (a more than 60-fold increase) pTyr at positions Y546 and Y584, sites known to positively regulate Shp-2 enzymatic activity (Figures 3A and 4A ; Table S1) (Zhang et al., 2015Zhang J. Zhang F. Niu R. Functions of Shp2 in cancer.J. Cell. Mol. Med. 2015; 19: 2075-2083Crossref PubMed Scopus (142) Google Scholar). We reasoned that Pdgfr-mediated activation of Shp-2 might be responsible for Y614 and Y615 dephosphorylation on Fak1 (Mañes et al., 1999Mañes S. Mira E. Gómez-Mouton C. Zhao Z.J. Lacalle R.A. Martínez-A C. Concerted activity of tyrosine phosphatase SHP-2 and focal adhesion kinase in regulation of cell motility.Mol. Cell. Biol. 1999; 19: 3125-3135Crossref PubMed Scopus (220) Google Scholar). We tested the impact of Shp-2 inhibition on Fak1 Y614 and Y615 dephosphorylation by a newly developed allosteric Shp-2 inhibitor SHP099 (Chen et al., 2016Chen Y.-N.P. LaMarche M.J. Chan H.M. Fekkes P. Garcia-Fortanet J. Acker M.G. Antonakos B. Chen C.H.-T. Chen Z. Cooke V.G. et al.Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases.Nature. 2016; 535: 148-152Crossref PubMed Scopus (479) Google Scholar) and observed that Shp-2 inhibition in Pdgf-ββ-treated cells led to increased Y614 and Y615 phosphorylation on Fak1 (Figure 4B). Although an increase of phosphorylation on Fak1 was also observed upon Fgf-2 treatment in the presence of the inhibitor, it was less pronounced relative to Pdgf-ββ stimulation (with SHP099), which inversed the phosphorylation dynamics on these two Fak1 pTyr sites. The results indicate that Shp-2 is an active player in Pdgfr signaling and directly affects Fak1 phosphorylation. We characterized the overall impact of Shp-2 activation in Pdgfr-mediated phosphosignaling response with a stable isotope labeling by amino acids in cell culture (SILAC)-based approach, in combination with phosphotyrosine peptide pull-down (Figure 5; Figure S6A) (Ong et al., 2002Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Crossref PubMed Scopus (4569) Google Scholar). Our findings revealed global changes in phosphorylation upon Pdgfr activation in Shp-2-inhibited cells (Figure S6B). Approximately 20% (1,428) of 6,884 quantified phosphorylation sites were statistically regulated more than 2-fold in the presence of SHP099 with Pdgf-ββ stimulation (based on Student’s t test, p < 0.05) (Table S2). Relative to serine and threonine phosphorylation sites, pTyr was mostly upregulated with Pdgf-ββ stimulation, alongside Shp-2 inhibitor (Figures S6B–S6E). Phosphotyrosine sites that were ≥2-fold upregulated (p < 0.05 based on Student’s t test) upon Pdgf-ββ stimulation in the presence of SHP099 were deemed Shp-2-putative substrates (Table S2). Our results revealed the negative regulation of Fak signaling (pY614/615) by Shp-2/Pdgfr was reversed in the presence of SHP099. Furthermore, pY421 on cortactin (Cttn), a known important substrate of Fak signaling that is suggested to modulate cytoskeletal organization and cell motility, displayed dramatic increase in phosphorylation upon Pdgf-ββ stimulation when cotreated with SHP099 (Figure S7A; Table S2) (Tomar et al., 2012Tomar A. Lawson C. Ghassemian M. Schlaepfer D.D. Cortactin as a target for FAK in the regulation of focal adhesion dynamics.PLoS ONE. 2012; 7: e44041Crossref PubMed Scopus (44) Google Scholar). We confirmed the dynamics of this key site on Cttn by using a site-specific antibody for pY421, which was abolished in the presence of Fak1 inhibitor PF-562271 (Figure 5B). In addition, Shp-2 and Fak1 were found to be constitutively associated and recruited to the Pdgfr signaling complex upon Pdgf-ββ stimulation (Figures S7B and S7C). Moreover, the results confirmed the specificity of Shp-2 inhibitor (SHP099) in inhibiting the Mapk/Erk pathway activation via significant downregulation of phospho-Erk1/2 activation sites pY185/205 (Figure 5) (Chen et al., 2016Chen Y.-N.P. LaMarche M.J. Chan H.M. Fekkes P. Garcia-Fortanet J. Acker M.G. Antonakos B. Chen C.H.-T. Chen Z. Cooke V.G. et al.Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases.Nature. 2016; 535: 148-152Crossref PubMed Scopus (479) Google Scholar). As a consequence, we identified many of the downregulated pSer sites that are predicted Erk1/2 substrates (Table S2). However, other signaling pathways were largely unaffected by Shp-2 inhibition, as determined by phosphorylation of prominent activation sites on Akt (pS473) and Stat (pY705), as well as pTyr sites on Plcg1 and Pik3c, which had unchanged or increased phosphorylation (Table S2). Our dataset demonstrates global upregulation of most phosphotyrosine signaling pathways by Shp-2 inhibition but accompanied by deactivation of the Mapk/Erk signaling cascade. Some of the most significantly upregulated pTyr sites with Shp-2 inhibition, alongside Pdgf-ββ stimulation, were found on proteins harboring guanosine triphosphatase (GTPase)-activating protein (GAP) domains (Figure 5A). We found significantly upregulated pTyr sites on GAP proteins spanning several GTPase families, such as Rasa1, Arhgap12, and Tbc1d10a (Figure 5A; Table S2). Increased levels of Rasa1, a Ras-specific GTPase, were observed to coimmunoprecipitate with Pdgfr upon Shp-2 inhibition and Pdgf-ββ stimulation (Figure 5C). pTyr on the crucial adaptor protein Grb2-associated binder 1 (Gab1) at pY628 was significantly downregulated, alongside markers of Erk/Mapk pathway activation (via phospho-Erk1/2 sites pY185/205) in the presence of Shp-2 inhibitor. Gab1-induced Erk activation after Met receptor activation has previously been shown to depend on Shp-2 activity (Maroun et al., 2003Maroun C.R. Naujokas M.A. Park M. Membrane targeting of Grb2-associated binder-1 (Gab1) scaffolding protein through Src myristoylation sequence substitutes for Gab1 pleckstrin homology domain and switches an epidermal growth factor response to an invasive morphogenic program.Mol. Biol. Cell. 2003; 14: 1691-1708Crossref PubMed Scopus (22) Google Scholar). Therefore, we hypothesized that this could be equivalent in Pdgfr-activated cells. We found Gab1 to significantly decrease its interaction with Pdgfr in the presence of Shp-2 inhibition (Figure 5C). Because Gab1 is known to be targeted to the plasma membrane upon RTK stimulation (Chang et al., 2015Chang C.-H. Chan P.-C. Li J.-R. Chen C.-J. Shieh J.-J. Fu Y.-C. Chen H.-C. Wu M.-J. Gab1 is essential for membrane translocation, activity and integrity of mTORCs after EGF stimulation in urothelial cell carcinoma.Oncotarget. 2015; 6: 1478-1489Crossref PubMed Scopus (9) Google Scholar), we observed an accumulation of Gab1 to the plasma membrane upon Pdgf-ββ stimulation (Figure 6). However, there was a significant reduction of Gab1 recruitment to the cellular plasma membrane after Pdgf-ββ stimulation in the presence of SHP099 (Figure 6B). The results indicate dynamic Shp-2-dependent regulation of key proteins, leading to changes in protein localization and pathway activation (Figure 6C). We next sought to determine the impact of Pdgfr- and Fgfr1-mediated signaling on cellular outcomes. Pdgf-ββ-induced cell migration and proliferation were both affected by Shp-2 inhibition, whereas Fgf-2-induced cellular responses were not (Figure 7). Specifically, Shp-2 inhibition, alongside Pdgf-ββ costimulation, of cells significantly reduced the rate at which cells migrated, while this was not observed with Fgf-2 stimulation in the presence of inhibitor (Figures 7A an 7B). The opposite trend was observed for cell proliferation in cells coexposed to SHP099 and Pdgf-ββ, displaying slightly higher proliferation rates comparable to Fgf-2 treatment. Conversely, SHP099 treatment, in combination with Fgf-2 stimulation, failed to affect the proliferation rate significantly (Figure 7C). Collectively, the results indicate an active role for Shp-2 in dictating Pdgf-ββ-mediated cellular migration, but not Fgf-2-dependent signaling and cellular responses. Tight regulation of the interplay among multiple cell signaling networks activated by RTKs is necessary to ensure normal cellular function. Identifying signaling proteins and quantifying their phosphorylation status directly by phosphoproteomics is emerging as an alternative to indirect approaches based on inferring cellular signaling network rewiring via genomic and transcriptomic studies (Yaffe, 2013Yaffe M.B. The scientific drunk and the lamppost: massive sequencing efforts in cancer discovery and treatment.Sci. Signal. 2013; 6: pe13Crossref PubMed Scopus (53) Google Scholar). In this work, we demonstrate proof-of-principle of detecting and quantifying phosphotyrosine-containing peptides simultaneously with pSer and pThr without additional affinity purification via cocktails of pan-anti-pTyr antibodies. This foundation proved to be essential in the global investigation of endogenous RTK signaling and activation in murine fibroblast cells. The dataset will serve as a valuable resource for further follow-up of novel proteins and phosphorylation sites that may be involved in regulating downstream RTK response. Because Pdgfr is a crucial RTK implicated in numerous cancers and a prominent target for therapy, understanding its downstream signaling components could provide invaluable insight for treatment. We conceptually demonstrated the potential of an antibody-free phosphoproteomic workflow by distinguishing the regulation of key downstream elements in Pdgfr signaling, such as Shp-2, Fak1, and Gab1, via pTyr. Although the relationships among these individual molecules have been well investigated as single- or two-component systems, our analysis pinpointed the interdepended regulation of each of these molecules simultaneously in an unbiased manner. The results highlight Shp-2 as a master regulator of downstream Pdgfr signaling while having modest to no influence on Fgfr signaling. Despite reduced cell migration upon Shp-2 inhibition, alongside Pdgfr activation, cell proliferation was surprisingly largely unaffected in our biological system. This could indicate different mechanisms of Mapk/Erk activation via activated Fgfr1 through distinct adaptor proteins such as Frs2. For instance, our results suggest that active Shp-2 is required for Gab1 recruitment to Pdgfr and the plasma membrane, which leads to full activation of the Erk/Mapk pathway. pTyr of Gab1 at pY628 has been suggested as a potential docking site for Shp-2 (Chan et al., 2010Chan P.-C. Sudhakar J.N. Lai C.-C. Chen H.-C. Differential phosphorylation of the docking protein Gab1 by c-Src and the hepatocyte growth factor receptor regulates different aspects of cell functions.Oncogene. 2010; 29: 698-710Crossref PubMed Scopus (18) Google Scholar, Cunnick et al., 2001Cunnick J.M. Mei L. Doupnik C.A. Wu J. Phosphotyrosines 627 and 659 of Gab1 constitute a bisphosphoryl tyrosine-based activation motif (BTAM) conferring binding and activation of SHP2.J. Biol. Chem. 2001; 276: 24380-24387Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar); however, it is possible that Shp-2 inhibition results in competitive Pdgfr interaction with an alternative protein superseding Gab1. It is conceivable that uncontrolled pTyr leads to the recruitment and localization of proteins and molecules that inhibit Erk1/2 activation. The significant increase in pTyr on several GAPs implies a crucial role in deactivating guanosine triphosphate (GTP)-bound Ras (active state), thus eliminating downstream activation of Erk via Ras/Raf/Mek. Unexpectedly, our results also indicate a potential role for GAPs, which have yet to be implicated with RTK signaling. Whether this results in reduced Gab1 pTyr and, as a consequence, reduced plasma membrane localization still needs to be determined. It is also plausible that additional molecular mechanisms and proteins could be involved in a synergistic or cumulative manner, leading to Mapk/Erk inhibition. Nonetheless, our dataset opens up the avenue for further examination of the role of several proteins that may regulate additional RTK signaling pathways in a similar manner and could serve as novel therapeutic targets. The importance of phosphotyrosine signaling in health and disease is unquestionable, but global and unbiased tyrosine phosphoproteomic investigations have so far been limited due to its low cellular abundance. We demonstrated the benefits of a global phosphoproteomic workflow for elucidating differential endogenous RTK signaling networks and expect similar workflows to be routinely applied directly toward analyzing signaling in tumor and cancer tissues. This provides opportunities to identify new biomarkers and drug candidates. As such, we hope that our datasets will serve as a beneficial foundation for further validation and mechanistic elucidation of several novel proteins that could be implicated in modulating downstream RTK signaling. 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• W2792103163 date "2018-03-01" @default.
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• W2792103163 title "Large-Scale Phosphoproteomics Reveals Shp-2 Phosphatase-Dependent Regulators of Pdgf Receptor Signaling" @default.
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• W2792103163 doi "https://doi.org/10.1016/j.celrep.2018.02.038" @default.
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